The p53 transcription factor, which is the focus of this work, is a potent tumor suppressor that works by activating expression of genes that control key cellular processes such as growth arrest and apoptosis. In order to activate its target genes, p53 interacts with a protein complex known as Mediator. Mediator helps regulate expression of nearly all protein-coding genes;thus, it lies at the heart of transcriptional regulation. Unlike most transcription factors, p53 interacts with Mediator via 2 different domains. Notably, using electron microscopy (EM) and single-particle reconstruction techniques, we have shown that each different p53 domain induces a dramatically different Mediator conformational state upon binding to the complex. The goal of this work is to clearly define the mechanistic role of each p53-Mediator interaction and how they may work together to regulate p53 activity. Experiments described in Aim 1 will use a reconstituted, human in vitro transcription system to examine how p53-induced structural changes affect the biochemical function of Mediator. In Aim 2 we will use immobilized template assays to explore whether p53-Mediator structural changes alter the assembly, composition, or stability of the transcriptional machinery (a.k.a. the Pre- Initiation Complex ). In Aim 3, we will combine cryo-EM studies with p53 crystal structure docking to reveal how different domains within the p53 tetramer work together to orchestrate such dramatic structural changes in Mediator. Taken together, these biochemical and structural studies will provide much-needed insight into the molecular mechanisms by which Mediator and p53 work together to control expression of key tumor suppressor genes.p53 is one of the most commonly mutated genes in human cancer. In order to properly function in preventing tumor formation, p53 must interact with a protein complex known as Mediator. Our work will define the fundamental molecular mechanisms by which p53 and Mediator work together to control expression of key anti-cancer genes. This will enhance our understanding of how p53 works to safeguard human cells against cancer;moreover, we anticipate that the information accumulated by our efforts will identify new strategies for controlling the anti-cancer activity of p53.